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Microbiology 154 (2008), 539-549; DOI  10.1099/mic.0.2007/010694-0
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Microbiology 154 (2008), 539-549; DOI  10.1099/mic.0.2007/010694-0
© 2008 Society for General Microbiology

Expression and immunogenicity of six putative variable surface proteins in Mycoplasma mycoides subsp. mycoides SC

Carl Hamsten1, Joakim Westberg2, Göran Bölske3, Roger Ayling4, Mathias Uhlén1 and Anja Persson1

1 Department of Proteomics, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, SE-106 91 Stockholm, Sweden
2 Department of Molecular Evolution, Evolutionary Biology Center, Uppsala University, SE-752 36 Uppsala, Sweden
3 Department of Bacteriology, National Veterinary Institute (SVA), SE-751 89 Uppsala, Sweden
4 The Mycoplasma Group, Veterinary Laboratories Agency (VLA), Addlestone, Surrey KT15 3NB, UK

Correspondence
Anja Persson
anja{at}biotech.kth.se

Variable surface protein Vmm and five Vmm-type proteins from Mycoplasma mycoides subsp. mycoides SC were analysed to determine whether these proteins are expressed in vivo in animals affected by contagious bovine pleuropneumonia (CBPP) and in vitro. Recombinant versions of these proteins were constructed and expressed in Escherichia coli after mutation of the TGA Trp codons to TGG. These proteins were then analysed by dot and Western blotting with sera from CBPP-affected cattle. Furthermore, affinity-purified polyclonal antibodies to the recombinant proteins were used in Western and colony blotting to look for expression of the putative Vmm-type proteins in cultured M. mycoides SC. This study demonstrates that immunoglobulins in CBPP sera recognize all putative Vmm-type proteins tested, indicating that these proteins or their homologues are expressed by mycoplasmas during natural infections. Vmm and one of the putative Vmm-type proteins showed variable expression in vitro.

Abbreviations: ABP, albumin binding protein; CBPP, contagious bovine pleuropneumonia; FBS, fetal bovine serum; HRP, horseradish peroxidase; IMAC, immobilized metal affinity chromatography; pAb, polyclonal antibody

A supplementary table listing the primers used in PCR, mutagenesis and DNA sequencing is available with the online version of this paper.






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